Nov 26, 2025
mcfDNA Sequencing from blood samples
- Krause Merlin1,
- Frederik Huhn1,
- M B2
- 1Center for Infectious Diseases and Infection Control, Jena University Hospital, Jena, Germany;
- 2UKJ
External link: https://nanoporetech.com/document/ligation-sequencing-v14-human-cfdna-multiplex-sqk-nbd114-24
Protocol Citation: Krause Merlin, Frederik Huhn, M B 2025. mcfDNA Sequencing from blood samples. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwqrr9vmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 02, 2025
Last Modified: November 26, 2025
Protocol Integer ID: 219362
Keywords: cfdna extraction, oxford nanopore ligation sequencing kit v14, cfdna, sensitive pathogen detection in clinical sample, mcfdna, free dna, sensitive pathogen detection, blood samples this protocol, dna repair, microbial cell, blood sample, clinical sample, downstream bioinformatic analysis, blood plasma
Disclaimer
This protocol corresponds to the protocol "multiplex sequencing of human cfDNA from blood using SQK-NBD114.24" provided by Oxford Nanopore.
Abstract
This protocol describes the preparation, library construction, and sequencing of human cell-free DNA (cfDNA) starting from blood plasma using the Oxford Nanopore Ligation Sequencing Kit V14 (SQK-NBD114.24) on GridION devices. The workflow comprises cfDNA extraction, DNA repair and end-prep, barcode and adapter ligation, and sequencing, followed by basecalling and downstream bioinformatic analysis. In our study, this workflow was applied to microbial cell-free DNA (mcfDNA) sequencing for rapid and sensitive pathogen detection in clinical samples.
Guidelines
Sample Collection & Handling:
- Tube Type: This protocol is optimized for Streck Cell-Free DNA BCT‱ tubes to minimize the lysis of human white blood cells. If using standard EDTA tubes, samples must be processed to plasma immediately (within 2–4 hours) to prevent genomic DNA contamination.
- Temperature: Do not freeze whole blood collected in Streck tubes; store and transport at room temperature (6 °C to 37 °C).
- Contamination Control: Microbial cell-free DNA (mcfDNA) analysis is highly sensitive to environmental contamination. Use aerosol-resistant (filter) pipette tips and nuclease-free water throughout the procedure.
Safety warnings
Biohazard / Biosafety Level 2:
Human blood and plasma should be treated as potentially infectious. Handle samples in a Class II biological safety cabinet (BSC) and wear appropriate personal protective equipment (PPE), including gloves, lab coat, and eye protection. Dispose of all waste in biohazard containers.
Ethics statement
Human Subjects Research:
Blood collection for this protocol requires prior approval by the users' Institutional Ethics Board or equivalent ethics committee. Ensure informed consent is obtained from all participants prior to sample collection in accordance with the Declaration of Helsinki.
Before start
Kit & Device Compatibility:
This protocol utilizes the QIAamp MinElute ccfDNA Midi Kit for extraction and the Ligation Sequencing Kit V14 (SQK-NBD114.24) for library prep.
Note on Volumes: While based on PromethION documentation, this specific protocol is adapted for GridION/MinION flow cells. Ensure priming and loading volumes in the final steps are appropriate for the specific flow cell type used (e.g., FLO-MIN114).
cfDNA extraction from patient blood
1h 15m 30s
Basis of this protocol: Kit for Isolation of mcfDNA
Draw approximately 10 mL Blood patient into two 10 mL Streck Cell-Free DNA BCT‱ tubes (Streck, USA) each and follow with step 2 and 3 as soon as possible.
4m
Centrifuge at 1900 x g for 10 minutes and distribute the supernatant into 2 ml Reaction tubes.1900 x g, 4°C, 00:10:00
10m
Centrifuge the plasma at 16000 x g for 10 minutes and collect the supernatant of each sample into one 15 ml tube.
The plasma can now be stored for up to a week at 4 °C until further processing.16000 x g, 4°C, 00:10:00
10m
To 1 ml Plasma add:
| Reagent | Volume [µl] | |
| Magentic Bead Suspension | 30 | |
| Proteinase K | 55 | |
| Bead Binding Buffer | 150 | |
| Total | 1235 |
3m
Optional: Spin down for 30 seconds at 200 x g in a swing out rotor centrifuge to remove any solution in the cap.
200 x g, Room temperature, 00:00:30
30s
Place tube into a magnetic rack for 00:02:00 or until the supernatant is clear before discarding the supernatant.
2m
Remove from magnetic rack and resuspend the beads in 200 µL Bead Elution Buffer . Pipette mix to rinse beads from the wall.
2m
Transfer the whole mixture into a 2 ml Bead Elution Tube.
1m
Incubate the Bead Elution Tube for 00:05:00 on a tube rotator mixer (Hula mixer) at room temperature.
5m
Place the Bead Elution Tube on a magnetic rack and let it stand for ≥ 00:01:00 until the solution is clear.
2m
Transfer the supernatant containing cfDNA into a new Bead Elution tube. Discard the bead pellet.
1m
Add 300 µL ACB Buffer to the Bead Elution tube containing the supernatant. Vortex to mix. Briefly centrifuge the tube to remove drops from inside the lid.
3m
Pipet the supernatant–ACB-Buffer mixture from the previous step into a QIAamp UCP MinElute column.
1m
Centrifuge for 1 min at 6,000 x g.
6000 x g, Room temperature, 00:01:00
1m
Place the QIAamp UCP MinElute column into a clean 2 ml collection tube. Discard the flow-through.
1m
Add 500 µL ACW2-Buffer to the QIAamp UCP MinElute column and centrifuge for 1 min at 6000 x g.
6000 x g, Room temperature, 00:01:00
1m
Place the QIAamp UCP MinElute column into a clean 2 ml collection tube. Discard the flow-through.
1m
Centrifuge the QIAamp UCP MinElute column at 20,000 x g for 3 min.
20000 x g, Room temperature, 00:03:00
3m
Place the QIAamp UCP MinElute column into a new 1.5 ml elution tube. Discard the 2 ml collection tube.
1m
Open the lid of the tube and incubate the assembly in a shaker for microcentrifuge tubes at 56°C for 3 min to dry the membrane completely.
00:03:00 56 °C
3m
Carefully pipet 23 µL ultra-clean water into the centre of the membrane. Close the lid and incubate at Room temperature for 00:15:00 .
15m
Centrifuge at 20,000 x g for 1 min to elute the DNA.
20000 x g, Room temperature, 00:01:00
1m
To maximise yield from the elution: Aspirate the eluate from the previous step and reload it onto the centre of the membrane. For this, carefully take off the QIAamp UCP MinElute column and after reloading, place it in the same 1.5 ml elution tube. Close the lid and incubate 00:01:00 at Room temperature .
1m
Centrifuge at 20,000 x g for 1 min to elute the DNA.
20000 x g, Room temperature, 00:01:00
1m
Quantify1 µL eluted sample using a Qubit fluorometer. Therefore add 1 µL eluted sample to 199 µL Qubit-Solution into a Qubit-Tube.
2m
The DNA can now be stored for up to a week at 4 °C until further processing.
Continue with the protocol when enough samples are collected so that the total amount of DNA for one GridION Flowcell is between 500 ng and 1000 ng to not load one Flowcell with a molar mass of more than 2000 fmol.
DNA repair and end-prep
2h 23m
Prepare the NEBNext FFPEDNA Repair Buffer v2, NEBNext FFPE DNA Repair Mix v2 and according to the protocol and place on ice.
2m
In each of the 0.2 ml thin-walled PCR tube containing your cfDNA samples, mix the following:
| Reagent | Volume [µl] | |
| cfDNA from the previous step | 20 | |
| NEBNext FFPE DNA Repair Buffer v2 | 3 | |
| NEBNext FFPE DNA Repair Mix v2 | 0.9 | |
| Total | 23.9 |
Use 20 µl of the DNA eluted in Step 30. Do not normalise samples by diluting with nuclease free water.
10m
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
2m
Using a thermal cycler with a heated lid set to 50 °C , incubate the reaction at 37 °C for 00:15:00 and hold at 4 °C .
15m
Remove the reaction from the thermal cycler and place the tube on ice.
30s
Keeping the tubes on ice, add 0.9 µL NEBNext Thermolabile Proteinase K directly to each of the repaired reaction mixtures.
2m
Mix by pipetting 10 times, followed by spinning down quickly to collect all liquid from the sides of the tube.
2m
Using a thermal cycler with a heated lid set to 75 °C , incubate at 37 °C for 00:15:00 and 65 °C for 00:05:00 , then hold at 4 °C .
20m
Remove the reaction from the thermal cycler and place the tube on ice.
30s
Keeping the tube on ice, add 1.3 µL NEBNext Ultra II End Prep Enzyme Mix directly to the reaction mixture for a total volume of 26.1 µl.
2m
Mix by pipetting 10 times, followed by spinning down quickly to collect all liquid from the sides of the tube.
2m
Using a thermal cycler with a heated lid set to 75 °C , incubate at 20 °C for 00:30:00 and 65 °C for 00:30:00 , then hold at 4 °C .
1h
Resuspend the AMPure XP Beads (AXP) by vortexing.
1m
Transfer each cfDNA sample into a separate clean 1.5 ml Eppendorf DNA LoBind tube.
2m
Add 80 µL resuspenden AMPure XP Beads (AXP) to each end-prep reaction and mix by flicking the tube.
2m
Incubate on a Hula mixer (rotator mixer) for 00:05:00 at room temperature.
5m
Prepare 5 mL 80 % fresh ethanol in nuclease-free water.
1m
Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.
1m
Keep the tube on the magnet and wash the beads with 200 µL 80 % Ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Note If the pellet was disturbed, wait for beads to pellet again before removing the ethanol.
1m
Repeat the previous step.
1m
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for 00:00:30 , but do not dry the pellet to the point of cracking.
2m
For each sample, remove the tube from the magnetic rack and resuspend the pellet in 10 µL nuclease-free water by flicking.
2m
Incubate for 00:02:00 at Room temperature .
2m
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 00:01:00 .
2m
For each sample, remove and retain 10 µL Eluate into a separate clean 1.5 ml Eppendorf DNA LoBind tube.
1m
Quantify 1 µL eluted sample using a Qubit fluorometer according to Step 26.
2m
Native barcode ligation
1h 23m
Prepare the Blunt/TA Ligase Master Mix according to the protocol and place on ice.
2m
Thaw the EDTA at Room temperature and mix by vortexing. Then spin down and place on ice.
2m
Thaw the Native Barcodes (NB01-24) at Room temperature . Briefly spin down, individually mix the barcodes required for your number of samples by pipetting, and place them on ice.
3m
Select a unique barcode for each sample to be run together on the same flow cell.
The number of samples barcoded and combined in one experiment depends on the total amount of DNA as described in Step 28.
In the 0.2 ml PCR-tubes containing your normalised sample inputs, add the reagents in the following order for each sample:
| Reagents | Volume [µl] | |
| End-prepped DNA | 7.5 | |
| Native Barcode (NB01-24) | 2.5 | |
| Blunt/TA Ligase Master Mix | 10 | |
| Total | 20 |
Use 7.5 µl of the DNA eluted in Step 53. Do not normalise samples by diluting with nuclease free water.
5m
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
2m
Incubate for 00:20:00 at Room temperature .
20m
Add the following volume of EDTA to each well and mix thoroughly by pipetting and spin down briefly.
| EDTA cap colour | Volume [µl] | |
| clear cap EDTA | 2 µl | |
| blue cap EDTA | 4 µl |
5m
Pool all the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube.
2m
Resuspend the AMPure XP Beads (AXP) by vortexing.
1m
Add 1.2X AMPure XP Beads (AXP) to the pooled reaction, and mix by pipetting.
The total volume of the pooled suspension is dependent on your number of samples.
Multiply your volume by 1.2 to get the right volume of AMPure XP Beads.
1m
Incubate on a Hula mixer for 00:10:00 at Room temperature .
10m
Prepare 2 mL fresh 80% ethanol in nuclease-free water.
2m
Spin down the sample and pellet on a magnet for 00:05:00 .
Pipette off the supernatant.
6m
Keep the tube on the magnetic rack and wash the beads with 700 µL 80 % Ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
2m
Repeat the previous step.
2m
Spin down and place the tube back on the magnetic rack. Pipette off any residual ethanol. Allow the pellet to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
2m
Remove the tube from the magnetic rack and resuspend the pellet in 32 µL nuclease-free water by gently flicking.
1m
Incubate for 00:10:00 at 37 °C . Every 00:02:00 , agitate the sample by gently flicking for 00:00:10 to encourage DNA elution.
11m
Pellet the beads on a magnetic rack until the eluate is clear and colourless.
1m
Remove and retain 32 µL eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
1m
Quantify 1 µl of eluted sample using a Qubit fluorometer according to step 26.
2m
Adapter ligation and clean-up
55m 5s
Prepare the NEBNext Quick Ligation Reaction Module (NEBNext Quick Ligation Reaction Buffer + Quick T4 DNA Ligase) according to the manufacturer's instructions, and place on ice.
Note: Do NOT vortex the Quick T4 DNA Ligase.
2m
Thaw the Native Adaper (NA) and Elution Buffer (EB) at Room temperature and place on ice.
2m
Spin down the reagent tubes for 00:00:05 .
5s
In a 1.5 ml Eppendorf LoBind tube, mix in the following order:
Between each addition, pipette mix 10 - 20 times.
| Reagents | Volume (µl) | |
| Pooled barcoded sample | 30 | |
| Native Adapter (NA) | 5 | |
| NEBNext Quick Ligation Reaction Buffer (5X) | 10 | |
| Quick T4 DNA Ligase | 5 | |
| Total | 50 |
10m
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
2m
Incubate the reaction for 00:20:00 at Room temperature .
20m
Resuspend the AMPure XP Beads (AXP) by vortexing.
1m
Add 60 µL (1.2x) resuspended AMPure XP Beads (AXP) to the reaction and mix by pipetting.
2m
Incubate on a Hula mixer for 00:10:00 at Room temperature .
10m
Spin down the sample and pellet on the magnetic rack. Keep the tube on the magnet and pipette off the supernatant.
3m
Quantify 1 µl of eluted sample using a Qubit fluorometer according to step 26.
3m
Note: The total amount of DNA should now be ≤430 ng to not exceed a molar mass of 2000 fmol with a DNA fragment length of 350 bp.
Priming and loading the GridION FlowCell
24m 40s
Thaw the Flow Cell Flush (FCF), BSA (50 mg/ml), Flow Cell Tether (FCT), Sequencing Buffer (SB) and Library Beads (LIB) at Room temperature . Place on ice.
4m
Before: Perform a Flowcell-Check
10m
Place your Flowcell in the GridIon-Device.
Open the MinKnow-Software and click on "Start". Go to "Flow Cell Check" and choose the position of your Flowcell and click "Start".
Prepare the Flowcell priming mix at room temperature. Mix by inverting.
| Reagents | Volume [µl] | |
| Flow Cell Flush (FCF) | 1.170 | |
| BSA (50 mg/ml) | 5 | |
| Flow Cell Tether (FCT) | 30 | |
| Total | 1.205 |
2m
To open the priming port of the flow cell, slide the port cover clockwise.
10s
Set a P1000 pipette to 200 µl. Insert the tip into the priming port. Turn the wheel of the pipette until the dial shows ~220-240 µl.
1m
Load 800 µL priming mix into the flow cell by using the priming port.
-> Dial the wheel of the pipette until a small drop of priming mix is visible on the pipette tip.
Incubate 00:05:00 . While Incubation proceed with step 93.
2m
Prepare the library in a new 1,5 ml Eppendorf tube. Thoroughly mix the Library Beads by pipetting.
| Reagents | Volume [µl] | |
| Sequencing Buffer (SB) | 37.5 | |
| Library Beads (LIB) | 25.5 | |
| DNA library | 12 | |
| Total | 75 |
3m
Gently open the SpotON sample port.
10s
Load 200 µL priming mix into the priming port.
1m
Mix the library by gently pipetting. In a dropwise fashin load 75 µL library into the flow cell via the SpotON port.
1m
Gently close the SpotON and priming port. Place the flow cell into the sequencing device. Start Sequencing.
20s
Protocol references
Oxford Nanopore Technologies. (2025). Ligation sequencing V14 – Human cfDNA multiplex (SQK-NBD114.24). https://nanoporetech.com/document/ligation-sequencing-v14-human-cfdna-multiplex-sqk-nbd114-24